BMP-11 compositions

ABSTRACT

Purified BMP-11 proteins and processes for producing them are disclosed. Recombinant DNA molecules encoding the BMP-11 proteins are also disclosed. The proteins may be useful in regulating follicle stimulating hormone, such as for contraception. In addition, the proteins may be useful for the induction of bone, cartilage and/or other connective tissue.

This application is a Continuation-in-part application of Ser. No.08/061,464 filed on May 12, 1993.

BACKGROUND OF THE INVENTION

The present invention relates to a novel family of purified proteinsdesignated BMP-11, DNA molecules encoding them, and processes forobtaining them. The inventors have previously designated the BMP-11proteins as Activin WC. The BMP-11 proteins may be useful to induce boneand/or cartilage formation and in wound healing and tissue repair, orfor augmenting the activity of other bone morphogenetic proteins. TheBMP-11 proteins may also be useful to regulate the production offollicle stimulating hormone, for contraception, to promote neuronalcell survival, to stimulate hematopoiesis, and to suppress thedevelopment of gonadal tumors.

U.S. Pat. No. 4,798,885 disclosed DNA encoding the prepro inhibin α andβ chains. U.S. Pat. No. 5,071,834 discloses pharmaceutical compositionsof activin with two beta_(B) chains formulated in a pharmaceuticallyacceptable carrier.

U.S. Pat. No. 5,102,807 discloses a purified inhibin protein whichsuppresses production of FSH without suppressing production ofluteinizing hormone.

SUMMARY OF THE INVENTION

BMP-11 protein is a member of the TGF-1 superfamily of proteins. TheTGF-β superfamily includes the family of proteins known as bonemorphogenetic proteins (BMPs), as well as a group of proteins that aretermed inhibin-β. As discussed further herein, when dimerized withanother BMP-11 (homodimer), BMP-11 protein is expected to demonstrateBMP-11 activity, as further described herein, as may be measured inaccordance with the assays described in the examples herein. Whendimerized as a heterodimer with inhibin-α proteins or with otherinhibin-t proteins, the inhibin-β/BMP-11 heterodimer is expected todemonstrate effects on the production of follicle stimulating hormone(FSH), as described further herein. It is further expected that, inhomodimeric form or in heterodimeric form with another member of thebone morphogenetic protein family, BMP-11 will exhibit BMP activity,i.e., the ability to induce the formation of bone, cartilage and/orother connective tissue. Thus, depending upon the environment of BMP-11,it may form dimers which will demonstrate either activin or inhibinactivity, or bone, cartilage and/or other connective tissue-inducingactivity. Accordingly, BMP-11 activity is defined as the ability toregulate the production of FSH in the assay described at Example 8herein, or the ability to induce the formation of bone, cartilage and/orother connective tissue in the assays described at Examples 5 to 7herein.

Proteins termed inhibins and activins are produced in the gonad andexist naturally in follicular fluid. These proteins act at the level ofthe anterior pituitary gland to inhibit (inhibins) or stimulate(activins) the release of follicle-stimulating hormone (FSH) [forreviews see, e.g., Ying, S.-Y., Endocr. Rev., 9:267-293 (1988) or Ling,N. et al, Vitamins and Hormones, 44:1-46 (Academic Press 1988)].Briefly, dimeric proteins comprised of one chain of inhibin a and onechain of inhibin β (β_(A) or β_(B)) are termed inhibins and arecharacterized by their ability to inhibit the release of folliclestimulating hormone (FSH), while other dimeric proteins comprised of twochains of inhibin β (β_(A) or β_(B)) are termed activins and arecharacterized by their ability to stimulate the release of folliclestimulating hormone (FSH) [see, e.g., Ling et al., Nature, 321:779-782(1986) or Vale, et al., Nature, 321:776-779 (1986) or Mason et al.,Nature, 318:659-663 (1985) or Forage et al., Proc. Natl. Acad. Sci. USA,83:3091-3095 (1986)].

It is recognized that FSH stimulates the development of ova in mammalianovaries (Ross et al., in Textbook of Endocrinology, ed. Williams, p. 355(−1981) and that excessive stimulation of the ovaries with FSH will leadto multiple ovulations. FSH is also important in testicular function.Thus, BMP-11, in heterodimers with a member of the inhibin a family, maybe useful as a contraceptive based on the ability of inhibins todecrease fertility in female mammals and decrease spermatogenesis inmale mammals. Administration of sufficient amounts of other inhibins caninduce infertility these mammals. BMP-11, as a homodimer or as aheterodimer with other protein subunits of the inhibin-β group, may beuseful as a fertility inducing therapeutic, based upon the ability ofactivin molecules in stimulating FSH release from cells of the anteriorpituitary. See, for example, U.S. Pat. No. 4,798,885. BMP-11 may also beuseful for advancement of the onset of fertility in sexually immaturemammals, so as to increase the lifetime reproductive performance ofdomestic animals such as cows, sheep and pigs. It is furthercontemplated that BMP-11 may be useful in promoting neuronal cellsurvival [see, e.g., Schubert et al., Nature, 344:868-870 (1990)],modulating hematopoiesis by inducing the differentiation of erythroidcells [see, e.g., Broxmeyer et al, Proc. Natl. Acad. Sci. USA,85:9052-9056 (1988) or Eto et al, Biochem. Biophys. Res. Comm.,142:1095-1103 (1987)], for suppressing the development of gonadal tumors[see, e.g., Matzuk et al., Nature, 360:313-319 (1992)] or for augmentingthe activity of bone morphogenetic proteins [see, e.g., Ogawa et al., J.Biol. Chem., 267:14233-14237 (1992)].

BMP-11 proteins may be further characterized by their ability tomodulate the release of follicle stimulating hormone (FSH) inestablished in vitro bioassays using rat anterior pituitary cells asdescribed [see, e.g., Vale et al, Endocrinology, 91:562-572 (1972); Linget al., Nature, 321:779-782 (1986) or Vale et al., Nature, 321:776-779(1986)]. It is contemplated that the BMP-11 protein of the invention,when composed as a homodimer or a heterodimer with other inhibin βchains will exhibit stimulatory effects on the release of folliclestimulating hormone (FSH) from anterior pituitary cells as described[Ling et al., Nature, 321:779-782 (1986) or Vale et al., Nature,321:776-779 (1986)].

Additionally, it is contemplated that the BMP-11 protein of theinvention, when composed as a heterodimer with the inhibin a chain, willinhibit the release of follicle stimulating hormone (FSH) from anteriorpituitary cells as described [see, e.g., Vale et al, Endocrinology,91:562-572 (1972). Therefore, depending on the particular composition,it is expected that the BMP-11 protein of the invention may havecontrasting and opposite effects on the release of follicle stimulatinghormone (FSH) from the anterior pituitary.

Activin A (the homodimeric composition of inhibin β_(A)) has been shownto have erythropoietic-stimulating activity [see e.g. Eto et al.,Biochem. Biophys. Res. Commun., 142:1095-1103 (1987) and Murata et al.,Proc. Natl. Acad. Sci. U.S.A., 85:2434-2438 (1988) and Yu et al.,Nature, 330:765-767 (1987)]. It is contemplated that the BMP-11 proteinof the invention has a similar erythropoietic-stimulating activity. Thisactivity of the BMP-11 protein may be further characterized by theability of the BMP-11 protein to demonstrate erythropoietin activity inthe biological assay performed using the human K-562 cell line asdescribed by [Lozzio et al., Blood, 45:321-334 (1975) and U.S. Pat. No.5,071,834].

The structures of several proteins, designated BMP-1 through BMP-9, havepreviously been elucidated. The unique inductive activities of theseproteins, along with their presence in bone, suggests that they areimportant regulators of bone repair processes, and may be involved inthe normal maintenance of bone tissue. The BMP-11 protein of the presentinvention is related to the above BMP proteins, and is expected to shareBMP activities such as the ability to induce bone, cartilage and/orother connective tissue, such as tendon or ligament, and wound healingactivities of the BMPs. In addition, it is expected that the proteins ofthe invention may act in concert with or perhaps synergistically withother related proteins and growth factors. Further therapeutic methodsand compositions of the invention therefore comprise a therapeuticamount of at least one BMP-11 protein of the invention with atherapeutic amount of at least one of the other BMP proteins disclosedin co-owned patents and applications described below. Such combinationsmay comprise separate molecules of the BMP proteins or heteromoleculescomprised of different BMP moieties. Further, BMP-11 proteins may becombined with other agents beneficial to the treatment of the boneand/or cartilage defect, wound, or tissue in question. These agentsinclude various growth factors such as epidermal growth factor (EGF),fibroblast growth factor (FGF), platelet derived growth factor (PDGF),transforming growth factors (TGF-α and TGF-β), and k-fibroblast growthfactor (kFGF), parathyroid hormone (PTH), leukemia inhibitory factor(LIF/HILDA/DIA), insulin-like growth factors (IGF-I and IGF-II).Portions of these agents may also be used in compositions of the presentinvention.

The bovine BMP-11 DNA sequence (SEQ ID NO: 1) and amino acid sequence(SEQ ID: 2) and human BMP-11 DNA sequence (SEQ ID NO:10) and amino acidsequence (SEQ ID NO:11) are set forth in the Sequence Listings herein.Activin proteins are capable of regulating the production offollicle-stimulating hormone (FSH), and thus BMP-11 may be useful as acontraceptive or a fertility inducing therapeutic. In homodimeric formor in heterodimers with proteins of the inhibin-β group, purified BMP-11protein is expected to demonstrate activin activity, and may be used tostimulate FSH. In addition, it is expected that the purified BMP-11protein may be useful for the induction of bone, cartilage and/or otherconnective tissue.

Bovine BMP-11 may be produced by culturing a cell transformed with a DNAsequence comprising nucleotide #375 to nucleotide #704 as shown in SEQID NO: 1 and recovering and purifying from the culture medium a proteincharacterized by the amino acid sequence comprising amino acid # 1 to #109 as shown in SEQ ID NO: 2 substantially free from other proteinaceousmaterials with which it is co-produced.

Human BMP-11 is expected to be homologous to bovine BMP-11. Theinvention, therefore, includes methods for obtaining the DNA sequencesencoding human BMP-11, the DNA sequences obtained by these methods, andthe human protein encoded by these DNA sequences. This method entailsutilizing the bovine BMP-11 nucleotide sequence or portions thereof todesign probes to screen libraries for the human gene or fragmentsthereof using standard techniques. A DNA sequence encoding part of thehuman BMP-11 protein (SEQ ID NO: 3) and the corresponding amino acidsequence (SEQ ID NO: 4) are set forth in the Sequence Listing. Thesesequences may also be used in order to design probes to obtain thecomplete human BMP-11 gene through standard techniques. Human BMP-11 maybe produced by culturing a cell transformed with the BMP-11 DNA sequenceand recovering and purifying BMP-11 from the culture medium. Thepurified expressed protein is substantially free from otherproteinaceous materials with which it is co-produced, as well as fromother contaminants.

The recovered purified protein is contemplated to demonstrate theability to regulate the production of FSH. The proteins of the inventionmay be further characterized by the ability to regulate the productionof follicle stimulating hormone (FSH) in established in vitro bioassaysusing rat anterior pituitary cells. BMP-11 proteins may also becharacterized by the ability to induce the formation of bone, cartilageand/or other connective tissue, for example, in the rat bone formationassay described below.

Another aspect of the invention provides pharmaceutical compositionscontaining a therapeutically effective amount of a BMP-11 protein in apharmaceutically acceptable vehicle or carrier. BMP-11 compositions ofthe invention may be useful for the regulation of follicle stimulatinghormone, and may be useful in contraception. Compositions of theinvention may further include at least one other therapeutically usefulagent such as the BMP proteins BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6and BMP-7, disclosed for instance in U.S. Pat. Nos. 5,108,922;5,013,649; 5,116,738; 5,106,748; 5,187,076; and 5,141,905; BMP-8,disclosed in PCT publication WO91/18098; and BMP-9, disclosed in PCTpublication WO93/00432, and BMP-10, disclosed in co-pending patentapplication Ser. No. 08/061,695, filed on May 12, 1993. The BMP-11compositions may also be useful for a number of uses involvingregulation of the production of follicle stimulating hormone, includingcontraception. These methods, according to the invention, entailadministering to a patient needing such treatment, an effective amountof BMP-11.

The compositions of the invention may comprise, in addition to a BMP-11protein, other members of the inhibin-t group of proteins or inhibin-αproteins, as well as other therapeutically useful agents includinggrowth factors such as epidermal growth factor (EGF), fibroblast growthfactor (FGF), transforming growth factor (TGF-α and TGF-β), andinsulin-like growth factor (IGF).

The BMP-11 compositions of the present invention may also be useful fortreating a number of bone and/or cartilage defects, periodontal diseaseand various types of wounds. These methods, according to the invention,entail administering to a patient needing such bone and/or cartilageformation wound healing or tissue repair, an effective amount of aBMP-11 protein. These methods may also entail the administration of aprotein of the invention in conjunction with at least one of the novelBMP proteins disclosed in the co-owned patents and applicationsdescribed above. In addition, these methods may also include theadministration of a BMP-11 protein with other growth factors includingEGF, FGF, TGF-α, TGF-β, and IGF.

Still a further aspect of the invention are DNA sequences coding forexpression of a BMP-11 protein. Such sequences include the sequence ofnucleotides in a 5′ to 3′ direction illustrated in SEQ ID NO: 1 or DNAsequences which hybridize under stringent conditions with the DNAsequence of SEQ ID NO: 1 and encode a protein having BMP-11 activity.Finally, allelic or other variations of the sequences of SEQ ID NO: 1,whether such nucleotide changes result in changes in the peptidesequence or not, are also included in the present invention.

Still a further aspect of the invention are DNA sequences coding forexpression of a BMP-11 protein. Such sequences include the sequence ofnucleotides in a 5′ to 3′ direction illustrated in SEQ ID NO: 1 or SEQID NO:10, and DNA sequences which, but for the degeneracy of the geneticcode, are identical to the DNA sequence of SEQ ID NO: 1 or SEQ ID NO:10,and encode the protein of SEQ ID NO: 2 or SEQ ID NO:11. Further includedin the present invention are DNA sequences which hybridize understringent conditions with the DNA sequence of SEQ ID NO: 1 or SEQ IDNO:10 and encode a protein having BMP-11 activity. Finally, allelic orother variations of the sequences of SEQ ID NO: 1 or SEQ ID NO: 10,whether such nucleotide changes result in changes in the peptidesequence or not, but where the peptide sequence still has BMP-11activity, are also included in the present invention.

A further aspect of the invention includes vectors comprising a DNAsequence as described above in operative association with an expressioncontrol sequence therefor. These vectors may be employed in a novelprocess for producing a BMP-11 protein of the invention in which a cellline transformed with a DNA sequence encoding a BMP-11 protein inoperative association with an expression control sequence therefor, iscultured in a suitable culture medium and a BMP-11 protein is recoveredand purified therefrom. This process may employ a number of known cellsboth prokaryotic and eukaryotic as host cells for expression of thepolypeptide.

The present invention also includes the use of the DNA sequences andvectors of the invention in gene therapy applications. In such use, thevectors may be transfected into the cells of a patient in vitro, and thecells may be re-introduced into a patient. Alternatively, the vectorsmay be introduced into a patient in vivo through targeted transfection.

Other aspects and advantages of the present invention will be apparentupon consideration of the following detailed description and preferredembodiments thereof.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a partial nucleotide sequence of the bovine BMP-11encoding the mature bovine BMP-11 polypeptide.

SEQ ID NO:2 is the amino acid sequence of a partial propeptide and thecomplete mature bovine BMP-11 polypeptide, encoded by SEQ ID NO:1.

SEQ ID NO:3 is a partial nucleotide sequence of human BMP-11.

SEQ ID NO:4 is a partial amino acid sequence for human BMP-11polypeptide encoded by SEQ ID NO:3.

SEQ ID NO:5 and 6 are primers to bovine BMP-11 used to isolate the humanBMP-11 or other BMP-11 proteins.

SEQ ID NO:7 is a DNA sequence that is inserted into pMT2 CXM to add anXhoI recognition site near the SV40 origin of replication.

SEQ ID NO:8 is a DNA sequence inserted into pMT21 to insert an XhoIrecognition site upstream from the DHFR gene.

SEQ ID NO:9 is a DNA sequence comprising a portion of the EMC virusleader sequence.

SEQ ID NO:10 is a DNA sequence encoding a partial propeptide and thecomplete mature human BMP-11 protein.

SEQ ID NO:11 is the amino acid sequence of a partial propeptide and thecomplete mature human BMP-11 protein encoded by SEQ ID NO:10.

DETAILED DESCRIPTION OF THE INVENTION

BMP-11

The bovine BMP-11 nucleotide sequence (SEQ ID NO: 1) and encoded aminoacid sequence (SEQ ID NO: 2) and human BMP-11 nucleotide sequence (SEQID NO:10) and encoded amino acid sequence (SEQ ID NO:11) are depicted inthe Sequence Listings herein. Purified bovine BMP-11 proteins of thepresent invention are produced by culturing a host cell transformed witha DNA sequence comprising the DNA coding sequence of SEQ ID NO: 1 fromnucleotide #375 to #704 or the DNA coding sequence of SEQ ID NO: 10 fromnucleotide #760 to #1086 and recovering and purifying from the culturemedium a protein which contains the amino acid sequence or asubstantially homologous sequence as represented by amino acids #1 to #109 of SEQ ID NO: 2 or amino acids 1 to 109 of SEQ ID NO: 11. Forproduction of BMP-11 proteins in mammalian cells, the DNA sequencefurther comprises a suitable propeptide linked in frame with the aboveDNA coding sequences f or BMP-11. The propeptide may be the nativepropeptide of BMP-11 or a propeptide from another member of the TGF-βsuperfamily

The human BMP-11 sequence of the present invention is obtained using thewhole or fragments of the bovine BMP-11 DNA sequence, or the partialhuman BMP-11 sequence of SEQ ID NO: 3 as a probe. Thus, the human BMP-11DNA sequence comprise the DNA sequence of nucleotides #28 to #185 of SEQID NO: 3. The human BMP-11 protein comprise the amino acid sequence ofamino acids #1 to #52 of SEQ ID NO: 4.

It is expected that BMP-11, as expressed by mammalian cells such as CHOcells, exists as a heterogeneous population of active species of BMP-11protein with varying N-termini. It is expected that active species willcomprise an amino acid sequence beginning at least with the cysteineresidue at amino acid #6 of SEQ ID NO:1 or SEQ ID NO:10, or further inthe N-terminal direction. Thus, it is expected that DNA sequencesencoding active BMP-11 proteins will comprise nucleotides #375 or #390to 701 of SEQ ID NO:1 or nucleotides #760 or #775 to #1086 of SEQ IDNO:110, and may comprise additional nucleotide sequence in the 5′direction of SEQ ID NO:1 or SEQ ID NO:10.

The N-terminus of human BMP-11 has been experimentally determined byexpression in E. coli to be as follows: [M]NLGLDXDEHSSE, wherein Xdesignates an amino acid residue with no clear signal, consistent with alocation of cysteine at that position. Thus, it is expected that thisspecies of BMP-11 will have an N-terminus at amino acid # 1 of SEQ IDNO:1 or SEQ. ID NO: 10, and DNA sequences encoding this species willcomprise nucleotides # 375 to # 701 of SEQ ID NO:1 (bovine) ornucleotides # 760 to 1086 of SEQ ID NO:10 (human). The apparentmolecular weight of human BMP-11 monomer was determined by SDS-PAGE tobe approximately 12 kd. The human BMP-11 protein exists as a clear,colorless solution in 0.1% trifluoroacetic acid.

The BMP-11 proteins recovered from the culture medium are purified byisolating them from other proteinaceous materials from which they areco-produced and from other contaminants present.

BMP-11 proteins may be characterized by the ability to LO regulate theproduction of FSH. BMP-11 proteins may further be characterized by theability to modulate the release of follicle stimulating hormone (FSH) inestablished in vitro bioassays using rat anterior pituitary cells asdescribed [see, e.g., Vale et al, Endocrinology, 91:562-572 (1972); Linget al., Nature, 321:779-782 (1986) or Vale et al., Nature, 321:776-779(1986)]. BMP-11 proteins may also be characterized by the ability toinduce the formation of bone, cartilage and/or other connective tissue.Such tissue-inducing activity of BMP-11 may further be characterized bythe ability to induce the formation of bone, cartilage and/or otherconnective tissue in the assays described in the examples below.

The BMP-11 proteins provided herein also include factors encoded by thesequences similar to those of SEQ ID NO: 1 or SEQ ID NO:10, but intowhich modifications are naturally provided (e.g. allelic variations inthe nucleotide sequence which may result in amino acid changes in thepolypeptide) or deliberately engineered. For example, syntheticpolypeptides may wholly or partially duplicate continuous sequences ofthe amino acid residues of SEQ ID NO:2 or SEQ ID NO:11. These sequences,by virtue of sharing primary, secondary, or tertiary structural andconformational characteristics with inhibin-β polypeptides of SEQ IDNO:2 or SEQ ID NO:11 may possess BMP-11 activity in common therewith.Thus, they may be employed as biologically active substitutes fornaturally-occurring BMP-11 polypeptides in therapeutic processes.

Other specific mutations of the sequences of BMP-11 proteins describedherein involve modifications of glycosylation sites. These modificationsmay involve O-linked or N-linked glycosylation sites. For instance, theabsence of glycosylation or only partial glycosylation results fromamino acid substitution or deletion at asparagine-linked glycosylationrecognition sites. The asparagine-linked glycosylation recognition sitescomprise tripeptide sequences which are specifically recognized byappropriate cellular glycosylation enzymes. These tripeptide sequencesare either asparagine-X-threonine or asparagine-X-serine, where X isusually any amino acid. A variety of amino acid substitutions ordeletions at one or both of the first or third amino acid positions of aglycosylation recognition site (and/or amino acid deletion at the secondposition) results in non-glycosylation at the modified tripeptidesequence. In addition, expression of the BMP-11 protein in bacterialcells results in non-glycosylated protein, without altering theglycosylation recognition sites.

The present invention also encompasses the novel DNA sequences, free ofassociation with DNA sequences encoding other proteinaceous materials,and coding for expression of BMP-11 proteins. These DNA sequencesinclude those depicted in SEQ ID NO:1 or SEQ ID NO:10 in a 5′ to 3′direction and those sequences which hybridize thereto under stringenthybridization conditions, for example 0.1×SSC, 0.1% SDS at 65° C. [see,Maniatis et al, Molecular Cloning (A Laboratory Manual), Cold SpringHarbor Laboratory (1982), pages 387 to 389] and encode a protein havingBMP-11 activity. These DNA sequences also include those which comprisethe DNA sequence of SEQ ID NO:3 and those which hybridize thereto understringent hybridization conditions and encode a protein having BMP-11activity.

Similarly, DNA sequences which code for BMP-11 proteins coded for by thesequences of SEQ ID NO:1 or SEQ ID NO:10, but which differ in codonsequence due to the degeneracies of the genetic code or allelicvariations (naturally-occurring base changes in the species populationwhich may or may not result in an amino acid change) also encode thenovel factors described herein. Variations in the DNA sequences of SEQID NO:1 or SEQ ID NO:10 which are caused by point mutations or byinduced modifications (including insertion, deletion, and substitution)to enhance the activity, half-life or production of the polypeptidesencoded are also encompassed in the invention.

Another aspect of the present invention provides a novel method forproducing BMP-11 proteins. The method of the present invention involvesculturing a suitable cell line, which has been transformed with a DNAsequence encoding a BMP-11 protein of the invention, under the controlof known regulatory sequences. The transformed host cells are culturedand the BMP-11 proteins recovered and purified from the culture medium.The purified proteins are substantially free from other proteins withwhich they are co-produced as well as from other contaminants.

Suitable cells or cell lines may be mammalian cells, such as Chinesehamster ovary cells (CHO). The selection of suitable mammalian hostcells and methods for transformation, culture, amplification, screening,product production and purification are known in the art. See, e.g.,Gething and Sambrook, Nature, 293:620-625 (1981), or alternatively,Kaufman et al, Mol. Cell. Biol., 5(7):1750-1759 (1985) or Howley et al,U.S. Pat. No. 4,419,446. Another suitable mammalian cell line, which isdescribed in the accompanying examples, is the monkey COS-1 cell line.The mammalian cell CV-1 may also be suitable.

Bacterial cells may also be suitable hosts. For example, the variousstrains of E. coli (e.g., HB101, MC1061) are well-known as host cells inthe field of biotechnology. Various strains of B. subtilis, Pseudomonas,other bacilli and the like may also be employed in this method.

Many strains of yeast cells known to those skilled in the art may alsobe available as host cells for expression of the polypeptides of thepresent invention. Additionally, where desired, insect cells may beutilized as host cells in the method of the present invention. See, e.g.Miller et al, Genetic Engineering, 8:277-298 (Plenum Press 1986) andreferences cited therein.

Another aspect of the present invention provides vectors for use in themethod of expression of these novel BMP-11 polypeptides. Preferably thevectors contain the full novel DNA sequences described above whichencode the novel factors of the invention. Additionally, the vectorscontain appropriate expression control sequences permitting expressionof the BMP-11 protein sequences. Alternatively, vectors incorporatingmodified sequences as described above are also embodiments of thepresent invention. Additionally, the sequence of SEQ ID NO:1 or SEQ IDNO:10 or other sequences encoding BMP-11 proteins could be manipulatedto express a mature BMP-11 by deleting BMP-11 encoding propeptidesequences and replacing them with sequences encoding the completepropeptides of other BMP proteins, activin proteins or other members ofthe TGF-β superfamily.

The vectors may be employed in the method of transforming cell lines andcontain selected regulatory sequences in operative association with theDNA coding sequences of the invention which are capable of directing thereplication and expression thereof in selected host cells. Regulatorysequences for such vectors are known to those skilled in the art and maybe selected depending upon the host cells. Such selection is routine anddoes not form part of the present invention.

For expression in mammalian host cells, the vector may comprise a codingsequence encoding a propeptide suitable for secretion of proteins by thehost cell linked in proper reading frame to the coding sequence formature BMP-11 protein. Suitable propeptide encoding sequences may beobtained from DNA encoding proteins of the TGF-β superfamily ofproteins, for example, including BMP-2 through BMP-9. For example, seeU.S. Pat. No. 5,168,150, the disclosure of which is hereby incorporatedby reference, in which a DNA encoding a precursor portion of a mammalianprotein other than BMP-2 is fused to the DNA encoding a mature BMP-2protein. Thus, the present invention includes chimeric DNA moleculescomprising a DNA sequence encoding a propeptide from a member of theTGF-β superfamily of proteins linked in correct reading frame to a DNAsequence encoding a BMP-11 polypeptide. The term “chimeric” is used tosignify that the propeptide originates from a different polypeptide thanBMP-11.

A protein of the present invention, which regulates the production ofFSH, has possible application in increasing fertility, when expressed ina composition as a homodimer or as a heterodimer with other proteins ofthe inhibin-β family. The proteins of the present invention may also beuseful for contraception, when expressed in a composition as aheterodimer with proteins of the inhibin-α family.

A protein of the present invention, which induces cartilage and/or boneformation in circumstances where bone is not normally formed, hasapplication in the healing of bone fractures and cartilage defects inhumans and other animals. Such a preparation employing a BMP-11 proteinmay have prophylactic use in closed as well as open fracture reductionand also in the improved fixation of artificial joints. De novo boneformation induced by an osteogenic agent contributes to the repair ofcongenital, trauma induced, or oncologic resection induced craniofacialdefects, and also is useful in cosmetic plastic surgery. A BMP-11protein may be used in the treatment of periodontal disease, and inother tooth repair processes. Such agents may provide an environment toattract bone-forming cells, stimulate growth of bone-forming cells orinduce differentiation of progenitors of bone-forming cells. BMP-11polypeptides of the invention may also be useful in the treatment ofosteoporosis. A variety of osteogenic, cartilage-inducing and boneinducing factors have been described. See, e.g., European patentapplications 148,155 and 169,016 for discussions thereof.

The proteins of the invention may also be used in wound healing andrelated tissue repair. The types of wounds include, but are not limitedto burns, incisions and ulcers. (See, e.g. PCT Publication WO84/01106for discussion of wound healing and related tissue repair).

A further aspect of the invention is a therapeutic method andcomposition for repairing fractures and other conditions related tocartilage and/or bone defects or periodontal diseases. The inventionfurther comprises therapeutic methods and compositions for wound healingand tissue repair. Such compositions comprise a therapeuticallyeffective amount of at least one of the BMP-11 proteins of the inventionin admixture with a pharmaceutically acceptable vehicle, carrier ormatrix.

Such a preparation employing a BMP-11 protein may also increase neuronalsurvival and therefore be useful in transplantation and treatment ofconditions exhibiting a decrease in neuronal survival.

It is expected that the BMP-11 proteins of the invention may act inconcert with or perhaps synergistically with other related proteins andgrowth factors. Further therapeutic methods and compositions of theinvention therefore comprise a therapeutic amount of at least one BMP-11protein of the invention with a therapeutic amount of at least one ofthe BMP proteins or other growth factors disclosed in co-owned patentsand applications described above. Such combinations may compriseseparate molecules or heteromolecules comprised of different moieties.For example, a method and composition of the invention may comprise adisulfide linked dimer comprising a BMP-11 protein subunit and a subunitfrom an inhibin-α protein, an inhibin-β protein or a BMP protein, suchas BMP-1 through BMP-10. The agents useful with BMP-11 may includevarious growth factors such as epidermal growth factor (EGF), plateletderived growth factor (PDGF), transforming growth factors (TGF-α andTGF-β), and insulin-like growth factor (IGF). Further therapeuticmethods and compositions of the invention comprise a therapeutic amountof at least one BMP-11 protein of the invention with a therapeuticamount of at least one of the BMP proteins disclosed in co-owned patentsand applications described above. Such combinations may compriseseparate molecules of the BMP proteins or heteromolecules comprised ofdifferent BMP moieties. For example, a method and composition of theinvention may comprise a disulfide linked dimer comprising a BMP-11protein subunit and a subunit from one of the “BMP” proteins describedabove. Thus, the present invention includes a purified BMP-11polypeptide which is a heterodimer wherein one subunit comprises atleast the amino acid sequence from amino acid #1 to amino acid #109 ofSEQ ID NO:2 or SEQ ID NO:11, and one subunit comprises an amino acidsequence for a bone morphogenetic protein selected from the groupconsisting of BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8 andBMP-9. A further embodiment may comprise a heterodimer of BMP-11moieties. Further, BMP-11 proteins may be combined with other agentsbeneficial to the treatment of the bone and/or cartilage defect, wound,or tissue in question. These agents include various growth factors suchas epidermal growth factor (EGF), fibroblast growth factor (FGF),platelet derived growth factor (PDGF), transforming growth factors(TGF-α and TGF-β), and k-fibroblast growth factor (kFGF), parathyroidhormone (PTH), leukemia inhibitory factor (LIF/HILDA/DIA), insulin-likegrowth factors (IGF-I and IGF-II). Portions of these agents may also beused in compositions of the present invention.

The BMP-11 proteins of the present invention may also be used incompositions combined with bone morphogenetic proteins. See for example,Ogawa et al., WO 92/14481 (1992); Ogawa et al., J. Biol. Chem.,267:14233-14237 (1992). The bone morphogenetic proteins useful in suchcompositions include BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7,disclosed for instance in U.S. Pat. Nos. 5,108,922; 5,013,649;5,116,738; 5,106,748; 5,187,076; and 5,141,905; BMP-8, disclosed in PCTpublication WO91/18098; and BMP-9, disclosed in PCT publicationWO93/00432; and BMP-10 disclosed in co-pending patent application Ser.No. 08/061,695, filed on May 12, 1993.

The preparation and formulation of such physiologically acceptableprotein compositions, having due regard to pH, isotonicity, stabilityand the like, is within the skill of the art. The therapeuticcompositions are also presently valuable for veterinary applications dueto the lack of species specificity in BMP and TGF proteins. Particularlydomestic animals and thoroughbred horses in addition to humans aredesired patients for such treatment with BMP-11 of the presentinvention.

The therapeutic method includes administering the composition topically,systemically, or locally as an implant or device. When administered, thetherapeutic composition for use in this invention is, of course, in apyrogen-free, physiologically acceptable form. Further, the compositionmay desirably be encapsulated or injected in a viscous form for deliveryto the site of bone, cartilage or tissue damage. Topical administrationmay be suitable for wound healing and tissue repair. Therapeuticallyuseful agents other than the BMP-11 proteins which may also optionallybe included in the composition as described above, may alternatively oradditionally, be administered simultaneously or sequentially with theBMP-11 composition in the methods of the invention.

Preferably for bone, cartilage or other connective tissue formation, thecomposition includes a matrix capable of delivering BMP-11 or other BMPproteins to the site of tissue damage in need of repair, providing astructure for the developing bone and cartilage and optimally capable ofbeing resorbed into the body. The matrix may provide slow release ofBMP-11 and/or other bone inductive protein, as well as properpresentation and appropriate environment for cellular infiltration. Suchmatrices may be formed of materials presently in use for other implantedmedical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the BMP-11compositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid andpolyanhydrides other potential materials are biodegradable andbiologically well defined, such as bone, tendon or dermal collagen.Further matrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are nonbiodegradable and chemicallydefined, such as sintered hydroxyapatite, bioglass, aluminates, or otherceramics. Matrices may be comprised of combinations of any of the abovementioned types of material, such as polylactic acid and hydroxyapatiteor collagen and tricalciumphosphate. The bioceramics may be altered incomposition, such as in calcium-aluminate-phosphate and processing toalter pore size, particle size, particle shape, and biodegradability.

Progress can be monitored by periodic assessment of bone growth and/orrepair. The progress can be monitored, for example, x-rays,histomorphometric determinations and tetracycline labeling.

The dosage regimen will be determined by the attending physicianconsidering various factors which modify the action of the BMP-11protein, e.g. the patient's age, sex, and diet, the severity of anyinfection, time of administration and other clinical factors. The dosagemay vary with the type of BMP protein or growth factor present in thecomposition. The dosage may also vary with the type of matrix used.

The following examples illustrate practice of the present invention inrecovering and characterizing bovine BMP-11 protein and employing it torecover the human and other BMP-11 proteins, obtaining the humanproteins and expressing the proteins via recombinant techniques.

EXAMPLE 1

Bovine BMP-11

800,000 recombinants of a bovine genomic library constructed in thevector λEMBL3 are plated at a density of 8000 recombinant bacteriophageplaques per plate on 100 plates. Duplicate nitrocellulose replicas ofthe recombinant bacteriophage plaques are made from these plates andamplified. A fragment of human BMP-7 DNA corresponding to nucleotides#1081 to #1403 (FIG. 4, U.S. Pat. No. 5,141,905) is ³²P-labelled by therandom priming procedure of Feinberg et al. [Anal. Biochem. 132: 6-13(1983)] and hybridized to one set of filters in standard hybridizationbuffer (SHB) (5×SSC, 0.170 SDS, 5× Denhardt's, 100 μg/ml Salmon spermDNA) at 60° C. for 2 to 3 days. The filters are washed under reducedstringency conditions (4×SSC, 0.1% SDS at 60° C.). Multiple positivelyhybridizing recombinants are noted. 52 positively hybridizingrecombinant bacteriophage plaques are selected and replated forsecondaries. Duplicate nitrocellulose replicas of the recombinantplaques are made from these 52 secondary plates and amplified. One setof nitrocellulose filters is hybridized to the human BMP-7 DNA probe asdescribed above and washed under the same reduced stringency conditions.The other set of filters is hybridized to a mixed BMP-5, BMP-6, andBMP-7 probe in SHB at 65° C. overnight and washed with a 0.1×SSC, 0.1%SDS at 65° C. (stringent hybridization and wash conditions). The mixedprobe consists of relatively equal amounts of ³²P-labelled DNA fragmentscomprising nucleotides #1452 to #2060 (FIG. 4, U.S. Pat. No. 5,106,748)of the human BMP-5 sequence, nucleotides #1395 to #1698 (FIG. 4, U.S.Pat. No. 5,187,076) of the human BMP-6 sequence, and nucleotides #1081to #1403 (FIG. 4, U.S. Pat. No. 5,141,905) of the human BMP-7 sequence.The BMP-5, BMP-6 and BMP-7 DNA fragments are ³²P-labelled by the randompriming procedure and equal numbers of counts per minute (cpms) of eachprobe are combined and added to the SHB containing the other set ofnitrocellulose filter replicas of the 52 secondary plates. 14recombinants, which hybridized positively to the human BMP-7 probe underthe reduced stringency conditions and exhibited weak or no hybridizationto the mixed BMP-5/6/7 probe under high stringency conditions, areselected for further analysis. All 14 recombinants which exhibit thesehybridization characteristics are plaque purified and bacteriophage DNAis prepared from each. The positively hybridizing region of one of the14 recombinants exhibiting the hybridization characteristics describedabove, designated λ7r-30, is localized to a 0.5 kb SacI restrictionfragment. This fragment is subcloned into a plasmid vector (pGEM-3) andDNA sequence analysis is performed. The partial DNA sequence (SEQUENCEID NO. 1) and derived amino acid sequence (SEQUENCE ID NO. 2) of cloneλ7r-30 are shown in the Sequence Listings.

The bacteriophage λ7r-30 has been deposited with the ATCC on Apr. 7,1993, and accorded the accession number ATCC 75439. This deposit meetsthe requirements of the Budapest Treaty of the International Recognitionof the Deposit of Microorganisms for the Purpose of Patent Proceduresand regulations thereunder.

This λ7r-30 clone encodes at least a portion of the bovine BMP-11protein of the present invention. The nucleotide sequence of cloneλ7r-30 contains an open reading frame of 456 nucleotides #246-701 of SEQID NO:1. The nucleotide sequence of #324 to #701 of SEQ ID NO:1 definesan open reading frame of 378 nucleotides, encoding at least 126 aminoacids of the C-terminal portion of a bovine BMP-11 protein, asdetermined by alignment to other BMP proteins and other proteins withinthe TGF-β family. The nucleotide sequence #246 to #323 defines an openreading frame contiguous with the sequence encoding the predicted 126amino acid BMP-11 peptide, however a reduced degree of amino acididentity of the peptide deduced from this region of DNA sequence (#246to #323) to other BMP proteins and other proteins of the TGF-β familyand the presence of multiple potential splice acceptor consensussequences make it difficult to define the 5′ limit of this exon of thebovine BMP-11 gene. The presence of an in-frame stop codon at nucleotidepositions #243 to #245 indicates that nucleotide sequence of cloneλ7r-30 contains at least one exon/intron boundary of the bovine BMP-11gene.

Based upon the knowledge of other proteins within the TGF-β family, itis predicted that the BMP-11 precursor polypeptide would be cleaved atthe multibasic sequence ARG-SER-ARG-ARG in agreement with a proposedconsensus proteolytic processing sequence of ARG-X-X-ARG. Cleavage ofthe BMP-11 precursor polypeptide is expected to generate a 109 aminoacid mature peptide beginning with the amino acid ASN at position #1.The processing of BMP-11 into the mature form is expected to involvedimerization and removal of the N-terminal region in a manner analogousto the processing of the related protein TGF-β [Gentry et al., Molec. &Cell. Biol., 8:4162 (1988); Derynck et al., Nature, 316:701(1985)].

It is contemplated therefore that the mature active species of BMP-11comprises a homodimer of two polypeptide subunits, each subunitcomprising amino acids # 1 to # 109 with a predicted molecular weight ofapproximately 12,000 daltons. Further active species are contemplatedcomprising amino acids #6 to #109, thereby including the first conservedcysteine residue. As with other members of the TGF-β family of proteins,the carboxy-terminal region of the BMP-11 protein exhibits greatersequence conservation than the more amino-terminal portion. The percentamino acid identity of the BMP-11 protein in the cysteine-richC-terminal domain (amino acids #6 to #109) to the corresponding regionof other proteins within the TGF-β family is as follows: BMP-2, 39%;BMP-3, 37%; BMP-4, 37%; BMP-5, 42%, BMP-6, 45%; BMP-7, 42%; BMP-8, 39%;BMP-9, 40%; Vg1, 39%; GDF-1, 34%; TGF-β1, 36%; TGF-β2, 38%; TGF-β3, 38%;inhibin β(B), 41%; inhibin β(A), 39%.

EXAMPLE 2

Human BMP-11

Bovine and human BMP-11 genes are presumed to be significantlyhomologous, therefore the bovine coding sequence or a portion thereof isused as a probe to screen a human genomic library or as a probe toidentify a human cell line or tissue which synthesizes the analogoushuman protein. A human genomic library, such as Stratagene catalog#944201, may be screened with such a probe, and presumptive positivesisolated and DNA sequence obtained. Evidence that this recombinantencodes a portion of the human BMP-11 relies on the bovine/human proteinand gene structure homologies.

Once a recombinant bacteriophage containing DNA encoding a portion ofthe human BMP-11 molecule is obtained, the human coding sequence can beused as a probe to identify a human cell line or tissue whichsynthesizes BMP-11 mRNA. Alternatively, the bovine BMP-11 codingsequence can be used as a probe to identify such human cell line ortissue. Briefly described, RNA is extracted from a selected cell ortissue source and either electrophoresed on a formaldehyde agarose geland transferred to nitrocellulose, or reacted with formaldehyde andspotted on nitrocellulose directly. The nitrocellulose is thenhybridized to a probe derived from a coding sequence of the bovine orhuman BMP-11. Alternatively, the bovine BMP-11 coding sequence is usedto design oligonucleotide primers which will specifically amplify aportion of the BMP-11 encoding sequence located in the region locatedbetween the primers utilized to perform the specific amplificationreaction. It is contemplated that bovine and human BMP-11 sequenceswould allow one to specifically amplify corresponding human BMP-11encoding sequences from mRNA, cDNA or genomic DNA templates. Once apositive source has been identified by one of the above describedmethods, mRNA is selected by oligo (dT) cellulose chromatography andcDNA is synthesized and cloned in λgt10 or other λ bacteriophage vectorsknown to those skilled in the art. (i.e. λZAP) by established techniques(Toole et al., supra). It is also possible to perform theoligonucleotide primer directed amplification reaction, described above,directly on a pre-established human cDNA or genomic library which hasbeen cloned into a λ bacteriophage vector. In such cases, a librarywhich yields a specifically amplified DNA product encoding a portion ofhuman BMP-11 protein could be screened directly, utilizing the fragmentof amplified BMP-11 encoding DNA as a probe.

Oligonucleotide primers designed on the basis of the DNA sequence of thebovine BMP-11 genomic clone λ7r-30 are predicted to allow the specificamplification of human BMP-11 encoding sequences. The followingoligonucleotide primer is designed on the basis of nucleotides #501 to#521 of the DNA sequence set forth in SEQ ID NO. 1 and synthesized on anautomated DNA synthesizer. Primer C: TAGTCTAGATGCTCCGGCCAGTGCGAGTAC

The first nine nucleotides of primer C (underlined) comprise therecognition sequence for the restriction endonuclease XbaI which can beutilized to facilitate the manipulation of a specifically amplified DNAsequence encoding the BMP-11 protein of the invention and are thus notderived from the DNA sequence presented in SEQ ID NO: 1.

The following oligonucleotide primer is designed on the basis ofnucleotides # 701 to # 678 of the DNA sequence set forth in SEQ ID NO. 1and synthesized on an automated DNA synthesizer: Primer D:TGCGGATCCGGAGCAGCCACAGCGATCCAC

The first nine nucleotides of primer D (underlined) comprise therecognition sequence for the restriction endonuclease BamHI which can beutilized to facilitate the manipulation of a specifically amplified DNAsequence encoding the BMP-11 protein of the invention and are thus notderived from the DNA sequence present in SEQ ID NO:1.

The standard nucleotide symbols in the above identified primers are asfollows: A, adenosine; C, cytosine, G, guanine; and T, thymine.

Primers C and D identified above are utilized as primers to allow theamplification of a specific nucleotide from human genomic DNA. Theamplification reaction is performed as follows:

Human genomic DNA (source: peripheral blood lymphocytes) is denatured at100° C. for five minutes and then chilled on ice prior to addition to areaction mixture containing 200 μM each deoxynucleotide triphosphates(dATP, dGTP, dCTP and dTTP) 10 mM Tris-HCl pH8.3, 50 mM KCl, 1.5 mMMgCl₂, 0.001% gelatin, 1.25 units Taq DNA polymerase, 100 pMoligonucleotide primer C and 100 pM oligonucleotide primer D. Thisreaction mixture is then subjected to thermal cycling in the followingmanner: 3 minutes at 94° C., 1 minute at 50° C., 1 minute at 72° C. forone cycle, then 1 minute at 94° C., 1 minute at 50° C., 1 minute at 72°C. for thirty-nine cycles.

The DNA which is specifically amplified by this reaction is separatedfrom the excess oligonucleotide primers C and D utilized to initiate theamplification by the use of a DNA purification resin based protocolunder the conditions suggested by the manufacturer. The resulting DNAproduct is digested with the restriction endonucleases XbaI and BamHI,phenol extracted, chloroform extracted. Buffer exchange and removal ofsmall fragments of DNA resulting from the XbaI/BaHI restriction digestis accomplished by dilution of the digested DNA product in 10 MmTris-Hcl pH8.0, 1 Mm EDTA followed by centrifugation through acentriconm 30 microconcentrator (W.R. Grace & Co., Beverly, Ma.; Product#4209). The resulting XbaI/BamHI digested amplified DNA product issubcloned into a plasmid vector (pBluescript) between the XbaI and BamHIrestriction sites of the polylinker region. DNA sequence analysis of theresulting subclones indicates that the specifically amplified DNAsequence product encodes a portion of the human BMP-11 protein of thisinvention. The DNA sequence (SEQ ID NO:3) and derived amino acidsequence (SEQ ID NO:4) of this specifically amplified DNA fragment areset forth in the Sequence Listings.

Nucleotides #1 to #27 of this sequence comprise a portion ofoligonucleotide primer C and nucleotides #186 to #213 comprise a portionof oligonucleotide primer D utilized to perform the specificamplification reaction. Due to the function of oligonucleotide primers Cand D (designed on the basis of bovine BMP-11 DNA sequence) ininitiating the amplification reaction, they may not correspond exactlyto the actual sequence encoding a human BMP-11 and are therefore nottranslated in the above amino acid sequence derivation. The DNAsequence, from nucleotide #28 to #185 of SEQ ID NO: 3, or portionsthereof, specifically amplified from the human genomic DNA template canbe utilized as a probe to identify additional human BMP-11 encodingsequences from human genomic or human cDNA libraries by standardhybridization/screening techniques known to those skilled in the art.

One million, two hundred thousand recombinants of a human fetal braincDNA library (Stratagene catalog # 936206) constructed in the vectorλZAPII are plated at a density of 24,000 recombinant bacteriophgeplaques per plate on 50 plates. Duplicate nitrocellulose replicas of therecombinant bacteriophage plaques are made from these plates. Anoligonucleotide probe designed on the basis of nucleotides #53-#82 ofSEQ ID NO:3 is synthesized on an automated DNA synthesizer. Thisoligonucleotide probe is radioactively labelled with γ³²P-ATP and ishybridized to both sets of the duplicate nitrocellulose replicas in SHBat 65° C. Nine positively hybridizing recombinants are noted. One of thepositively hybridizing recombinants, named λFB30.5, is plaque purified.Bacteriophage plate stocks of the purified λFB30.5 cDNA clone areprepared and bacteriophage DNA is isolated. A bacterial plasmid namedFB30.5, generated by the in vivo excision protocol described by thesupplier (Stratagene) and containing the entire insert of the λFB30.5bacteriophage cDNA clone, has been deposited with the ATCC, 12301Parklawn Drive, Rockville, Md. USA under the requirements of theBudapest Treaty and designated as ATCC # ______. A portion of the DNAsequence of clone FB30.5 is set forth in SEQ ID NO: 10.

One million recombinants of a human genomic library (Stratagene Catalog# 944201) constructed in the vector λFIX are plated at a density of20,000 recombinant bacteriophge plaques per plate on 50 plates.Duplicate nitrocellulose replicas of the recombinant bacteriophageplaques are made from these plates. An oligonucleotide probe designed onthe basis of nucloetides #57-#86 of SEQ ID NO:10, with the exception ofan inadvertent substitution of CAC for GCG at nucleotides #59-#61 of SEQID NO:10, is synthesized on an automated DNA synthesizer. Thisoligonucleotide probe is radioactively labelled with γ³²P-ATP and ishybridized to both sets of the duplicate nitrocellulose replicas in SHBat 65° C. Five positively hybridizing recombinants are noted. One of thepositively hybridizing recombinants, named 30GEN.4, is plaque purified.Bacteriophage plate stocks of the purified 30GEN.4 genomic clone areprepared and bacteriophage DNA is isolated. A bacteriophage stock ofthis genomic clone has been deposited with the ATCC, 12301 ParklawnDrive, Rockville, Md. USA under the requirements of the Budapest Treatyand designated as ATCC # ______. A portion of the DNA sequence of clone30GEN.4 is set forth in SEQ ID NO: 10. A portion of the DNA sequence ofthe genomic clone 30GEN.4 was determined to be identical to a portion ofthe DNA sequence of the cDNA clone FB30.5. The extent of this overlap(nucleotides #1-#198) of SEQ ID NO:10 were used as a basis to compilethe partial coding sequence of the BMP-10 protein. The genomic clone30GEN.4 is expected to contain additional 5′ coding sequences of thehuman BMP-11 protein which are expected to encode the remainder of theBMP-11 precursor polypeptide, including the initiator methionine. Thepartial sequence of human BMP-11 is presented in SEQ ID NO:10 and itshould be noted that nucleotides #1-198 have been determined to bepresent in both the 30GEN.4 genomic clone and the FB30.5 cDNA clonewhile nucleotides #199-#1270 are derived entirely from the cDNA cloneFB30.5. SEQ ID NO:10 predicts a human BMP11 precursor protein of atleast 362 amino acids. Based on the knowledge of other BMPs and otherproteins within the TGF-β family, it is predicted that the precursorpolypeptide would be cleaved at the multibasic sequence ARG-SER-ARG-ARG(amino acids #-4 through #-1 of SEQ ID NO:11) in agreement with theproposed consensus proteolytic processing sequence ARG-X-X-ARG. Cleavageof the human BMP-11 precursor polypeptide at this location wouldgenerate a 109 amino acid mature peptide beginning with the amino acidASN at position #1 of SEQ ID NO:11. The processing of human BMP-11 intothe mature form is expected to involve dimerization and removal of theN-terminal region in a manner analogous to the procesing of the relatedprotein TGF-β [L. E. Gentry, et al. Molec. & Cell. Biol. 8:4162 (1988);R. Derynck, et al., Nature 316:701 (1985). It is contemplated that themature active species of human BMP-11 comprises a homodimer of twopolypeptide subunits, each subunit comprising amino acids #1-#108 of SEQID NO:11, with a predicted molecular weight of 12,000 daltons. Furtheractive species are contemplated comprising amino acids #7-#108 of SEQ IDNO:11, thereby including the first conserved cysteine residue.Heterodimeric molecules comprising one subunit of BMP-11 and anothersubunit of another member of the BMP/TGF-β superfamily are alsocontemplated.

EXAMPLE 3

Expression of BMP-11

In order to produce bovine, human or other mammalian BMP-11 proteins,the DNA encoding it is transferred into an appropriate expression vectorand introduced into mammalian cells or other preferred eukaryotic orprokaryotic hosts by conventional genetic engineering techniques. Thepreferred expression system for biologically active recombinant humanBMP-11 is contemplated to be stably transformed mammalian cells.

One skilled in the art can construct mammalian expression vectors byemploying the sequence of SEQ ID NO:1 or SEQ ID NO:10, or other DNAsequences encoding BMP-11 proteins or other modified sequences and knownvectors, such as pCD [Okayama et al., Mol. Cell Biol., 2:161-170(1982)], pJL3, pJL4 (Gough et al., EMBO J., 4:645-653 (1985)] and pMT2CXM.

The mammalian expression vector pMT2 CXM is a derivative of p91023(b)(Wong et al., Science 228:810-815, 1985) differing from the latter inthat it contains the ampicillin resistance gene in place of thetetracycline resistance gene and further contains a XhoI site forinsertion of cDNA clones. The functional elements of pMT2 CXM have beendescribed (Kaufman, R. J., 1985, Proc. Natl. Acad. Sci. USA 82:689-693)and include the adenovirus VA genes, the SV40 origin of replicationincluding the 72 bp enhancer, the adenovirus major late promoterincluding a 5′ splice site and the majority of the adenovirus tripartiteleader sequence present on adenovirus late mRNAs, a 3′ splice acceptorsite, a DHFR insert, the SV40 early polyadenylation site (SV40), andpBR322 sequences needed for propagation in E. coli.

Plasmid pMT2 CXM is obtained by EcoRI digestion of pMT2-VWF, which hasbeen deposited with the American Type Culture Collection (ATCC),Rockville, Md. (USA) under accession number ATCC 67122. EcoRI digestionexcises the cDNA insert present in pMT2-VWF, yielding pMT2 in linearform which can be ligated and used to transform E. coli HB 101 or DH-5to ampicillin resistance. Plasmid pMT2 DNA can be prepared byconventional methods. pMT2 CXM is then constructed using loopout/inmutagenesis (Morinaga, et al., Biotechnology 84: 636 (1984). Thisremoves bases 1075 to 1145 relative to the Hind III site near the SV40origin of replication and enhancer sequences of pMT2. In addition itinserts the following sequence: 5′ PO-CATGGGCAGCTCGAG-3′at nucleotide 1145. This sequence contains the recognition site for therestriction endonuclease Xho I. A derivative of pMT2CXM, termed pMT23,contains recognition sites for the restriction endonucleases PstI, EcoRI, SalI and XhoI. Plasmid pMT2 CXM and pMT23 DNA may be prepared byconventional methods.

pEMC2β1 derived from pMT21 may also be suitable in practice of theinvention. pMT21 is derived from pMT2 which is derived from pMT2-VWF. Asdescribed above EcoRI digestion excises the cDNA insert present inpMT-VWF, yielding pMT2 in linear form which can be ligated and used totransform E. Coli HB 101 or DH-5 to ampicillin resistance. Plasmid pMT2DNA can be prepared by conventional methods.

pMT21 is derived from pMT2 through the following two modifications.First, 76 bp of the 5′ untranslated region of the DHFR cDNA including astretch of 19 G residues from G/C tailing for cDNA cloning is deleted.In this process, a XhoI site is inserted to obtain the followingsequence immediately upstream from DHFR:5′-CTGCAGGCGAGCCTGAATTCCTCGAGCCATCATG-3′                           PstI        Eco RI XhoI

Second, a unique ClaI site is introduced by digestion with EcoRV andXbaI, treatment with Klenow fragment of DNA polymerase I, and ligationto a ClaI linker (CATCGATG). This deletes a 250 bp segment from theadenovirus associated RNA (VAI) region but does not interfere with VAIRNA gene expression or function. pMT21 is digested with EcoRI and XhoI,and used to derive the vector pEMC2B1.

A portion of the EMCV leader is obtained from pMT2-ECAT1 [S. K. Jung, etal, J. Virol 63:1651-1660 (1989)] by digestion with Eco RI and PstI,resulting in a 2752 bp fragment. This fragment is digested with TaqIyielding an Eco RI-TaqI fragment of 508 bp which is purified byelectrophoresis on low melting agarose gel. A 68 bp adapter and itscomplementary strand are synthesized with a 5′ TaqI protruding end and a3′ XhoI protruding end which has the following sequence:5′-CGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTT    TaqITCCTTTGAAAAACACGATTGC-3′                     XhoIThis sequence matches the EMC virus leader sequence from nucleotide 763to 827. It also changes the ATG at position 10 within the EMC virusleader to an ATT and is followed by a XhoI site. A three way ligation ofthe pMT21 Eco RI-XhoI fragment, the EMC virus EcoRI-TaqI fragment, andthe 68 bpoligonucleotide adapter TaqI-XhoI adapter resulting in the vectorpEMC2β1.

This vector contains the SV40 origin of replication and enhancer, theadenovirus major late promoter, a cDNA copy of the majority of theadenovirus tripartite leader sequence, a small hybrid interveningsequence, an SV40 polyadenylation signal and the adenovirus VA I gene,DHFR and β-lactamase markers and an EMC sequence, in appropriaterelationships to direct the high level expression of the desired cDNA inmammalian cells.

The construction of vectors may involve modification of the BMP-11 DNAsequences. For instance, BMP-11 cDNA can be modified by removing thenon-coding nucleotides on the 5′ and 3′ ends of the coding region. Thedeleted non-coding nucleotides may or may not be replaced by othersequences known to be beneficial for expression. These vectors aretransformed into appropriate host cells for expression of BMP-11proteins. Additionally, the sequence of SEQ ID NO:1 or SEQ ID NO:10 orother sequences encoding BMP-11 proteins could be manipulated to expressa mature BMP-11 by deleting BMP-11 encoding propeptide sequences andreplacing them with sequences encoding the complete propeptides of otherBMP proteins, activin proteins or other members of the TGF-βsuperfamily.

One skilled in the art can manipulate the sequences of SEQ ID NO:1 orSEQ ID.NO:10 by eliminating or replacing the mammalian regulatorysequences flanking the coding sequence with bacterial sequences tocreate bacterial vectors for intracellular or extracellular expressionby bacterial cells. For example, the coding sequences could be furthermanipulated (e.g. ligated to other known linkers or modified by deletingnon-coding sequences therefrom or altering nucleotides therein by otherknown techniques). The modified BMP-11 coding sequence could then beinserted into a known bacterial vector using procedures such asdescribed in T. Taniguchi et al., Proc. Natl. Acad. Sci. USA,77:5230-5233 (1980). This exemplary bacterial vector could then betransformed into bacterial host cells and a BMP-11 protein expressedthereby. For a strategy for producing extracellular expression of BMP-11proteins in bacterial cells, see, e.g. European patent application EPA177,343.

Similar manipulations can be performed for the construction of an insectvector [See, e.g. procedures described in published European patentapplication 155,476] for expression in insect cells. A yeast vectorcould also be constructed employing yeast regulatory sequences forintracellular or extracellular expression of the factors of the presentinvention by yeast cells. [See, e.g., procedures described in publishedPCT application WO86/00639 and European patent application EPA 123,289].

A method for producing high levels of a BMP-11 protein of the inventionin mammalian cells may involve the construction of cells containingmultiple copies of the heterologous BMP-11 gene. The heterologous geneis linked to an amplifiable marker, e.g. the dihydrofolate reductase(DHFR) gene for which cells containing increased gene copies can beselected for propagation in increasing concentrations of methotrexate(MTX) according to the procedures of Kaufman and Sharp, J. Mol. Biol.,159:601-629 (1982). This approach can be employed with a number ofdifferent cell types.

For example, a plasmid containing a DNA sequence for a BMP-11 of theinvention in operative association with other plasmid sequences enablingexpression thereof and the DHFR expression plasmid pAdA26SV(A)₃ [Kaufmanand Sharp, Mol. Cell. Biol., 2:1304 (1982)] can be co-introduced intoDHFR-deficient CHO cells, DUKX-BII, by various methods including calciumphosphate coprecipitation and transfection, electroporation orprotoplast fusion. DHFR expressing transformants are selected for growthin alpha media with dialyzed fetal calf serum, and subsequently selectedfor amplification by growth in increasing concentrations of MTX (e.g.sequential steps in 0.02, 0.2, 1.0 and 5 uM MTX) as described in Kaufmanet al., Mol Cell Biol., 5:1750 (1983). Transformants are cloned, andbiologically active BMP-11 expression is monitored by one or more of theBMP-11 activity assays described in Examples 5 to 8 below. BMP-11expression should increase with increasing levels of MTX resistance.BMP-11 polypeptides are characterized using standard techniques known inthe art such as pulse labeling with [³⁵S] methionine or cysteine andpolyacrylamide gel electrophoresis. Similar procedures can be followedto produce other related BMP-11 proteins.

EXAMPLE 4

Biological Activity of Expressed BMP-11

To measure the biological activity of the expressed BMP-11 proteinsobtained in Example 3 above, the proteins are recovered from the cellculture and purified by isolating the BMP-11 proteins from otherproteinaceous materials with which they are co-produced as well as fromother contaminants. The purified protein may be assayed in accordancewith the assays for BMP-11 activity described in Examples 5 to 8 below.

EXAMPLE 5

W-20 BIOASSAYS

A. Description of W-20 Cells

Use of the W-20 bone marrow stromal cells as an indicator cell line isbased upon the conversion of these cells to osteoblast-like cells aftertreatment with a BMP protein [Thies et al, Journal of Bone and MineralResearch, 5:305 (1990); and Thies et al, Endocrinology, 130:1318(1992)]. Specifically, W-20 cells are a clonal bone marrow stromal cellline derived from adult mice by researchers in the laboratory of Dr. D.Nathan, Children's Hospital, Boston, Mass. Treatment of W-20 cells withcertain BMP proteins results in (1) increased alkaline phosphataseproduction, (2) induction of PTH stimulated cAMP, and (3) induction ofosteocalcin synthesis by the cells. While (1) and (2) representcharacteristics associated with the osteoblast phenotype, the ability tosynthesize osteocalcin is a phenotypic property only displayed by matureosteoblasts. Furthermore, to date we have observed conversion of W-20stromal cells to osteoblast-like cells only upon treatment with BMPs. Inthis manner, the in vitro activities displayed by BMP treated W-20 cellscorrelate with the in vivo bone forming activity known for BMPs.

Below two in vitro assays useful in comparison of BMP activities ofnovel osteoinductive molecules are described.

B. W-20 Alkaline Phosphatase Assay Protocol

W-20 cells are plated into 96 well tissue culture plates at a density of10,000 cells per well in 200 μl of media (DME with 10% heat inactivatedfetal calf serum, 2 mM glutamine and 100 Units/ml penicillin +100 μg/mlstreptomycin. The cells are allowed to attach overnight in a 95% air, 5%CO₂ incubator at 37° C.

The 200 μl of media is removed from each well with a multichannelpipettor and replaced with an equal volume of test sample delivered inDME with 10% heat inactivated fetal calf serum, 2 mM glutamine and 1%penicillin-streptomycin. Test substances are assayed in triplicate.

The test samples and standards are allowed a 24 hour incubation periodwith the W-20 indicator cells. After the 24 hours, plates are removedfrom the 37° C. incubator and the test media are removed from the cells.

The W-20 cell layers are washed 3 times with 200 μl per well ofcalcium/magnesium free phosphate buffered saline and these washes arediscarded.

50 μl of glass distilled water is added to each well and the assayplates are then placed on a dry ice/ethanol bath for quick freezing.Once frozen, the assay plates are removed from the dry ice/ethanol bathand thawed at 37° C. This step is repeated 2 more times for a total of 3freeze-thaw procedures. Once complete, the membrane bound alkalinephosphatase is available for measurement.

50 μl of assay mix (50 mM glycine, 0.05% Triton X-100, 4 mM MgCl₂, 5 mMp-nitrophenol phosphate, pH=10.3) is added to each assay well and theassay plates are then incubated for 30 minutes at 37° C. in a shakingwaterbath at 60 oscillations per minute.

At the end of the 30 minute incubation, the reaction is stopped byadding 100 μl of 0.2 N NaOH to each well and placing the assay plates onice.

The spectrophotometric absorbance for each well is read at a wavelengthof 405 nanometers. These values are then compared to known standards togive an estimate of the alkaline phosphatase activity in each sample.For example, using known amounts of p-nitrophenol phosphate, absorbancevalues are generated. This is shown in Table I. TABLE I AbsorbanceValues for Known Standards of P-Nitrophenol Phosphate P-nitrophenolphosphate umoles Mean absorbance (405 nm) 0.000 0 0.006 0.261 +/− .0240.012 0.521 +/− .031 0.018 0.797 +/− .063 0.024 1.074 +/− .061 0.0301.305 +/− .083

Absorbance values for known amounts of BMPs can be determined andconverted to μmoles of p-nitrophenol phosphate cleaved per unit time asshown in Table II. TABLE II Alkaline Phosphatase Values for W-20 CellsTreating with BMP-2 BMP-2 concentration Absorbance Reading umolessubstrate ng/ml 405 nmeters per hour 0 0.645 0.024 1.56 0.696 0.026 3.120.765 0.029 6.25 0.923 0.036 12.50 1.121 0.044 25.0 1.457 0.058 50.01.662 0.067 100.0 1.977 0.080

These values are then used to compare the activities of known amounts ofBMP-11 to BMP-2.

C. Osteocalcin RIA Protocol

W-20 cells are plated at 10⁶ cells per well in 24 well multiwell tissueculture dishes in 2 mls of DME containing 10% heat inactivated fetalcalf serum, 2 mM glutamine. The cells are allowed to attach overnight inan atmosphere of 95% air 5% CO₂ at 37° C.

The next day the medium is changed to DME containing 10% fetal calfserum, 2 mM glutamine and the test substance in a total volume of 2 ml.Each test substance is administered to triplicate wells. The testsubstances are incubated with the W-20 cells for a total of 96 hourswith replacement at 48 hours by the same test medias.

At the end of 96 hours, 50 μl of the test media is removed from eachwell and assayed for osteocalcin production using a radioimmunoassay formouse osteocalcin. The details of the assay are described in the kitmanufactured by Biomedical Technologies Inc., 378 Page Street,Stoughton, Mass. 02072. Reagents for the assay are found as productnumbers BT-431 (mouse osteocalcin standard), BT-432 (Goat anti-mouseOsteocalcin), BT-431R (iodinated mouse osteocalcin), BT-415 (normal goatserum) and BT-414 (donkey anti goat IgG). The RIA for osteocalcinsynthesized by W-20 cells in response to BMP treatment is carried out asdescribed in the protocol provided by the manufacturer.

The values obtained for the test samples are compared to values forknown standards of mouse osteocalcin and to the amount of osteocalcinproduced by W-20 cells in response to challenge with known amounts ofBMP-2. The values for BMP-2 induced osteocalcin synthesis by W-20 cellsis shown in Table III. TABLE III Osteocalcin Synthesis by W-20 CellsBMP-2 Concentration Osteocalcin Synthesis ng/ml ng/well 0 0.8 2 0.9 40.8 8 2.2 16 2.7 31 3.2 62 5.1 125 6.5 250 8.2 500 9.4 1000 10.0

EXAMPLE 6

Rosen-Modified Sampath-Reddi Assay.

A modified version of the rat bone formation assay described in Sampathand Reddi, Proc. Natl. Acad. Sci. USA, 80:6591-6595 (1983) is used toevaluate bone, cartilage and/or other connective tissue inductiveactivity of BMP-11 proteins. This modified assay is herein called theRosen-modified Sampath-Reddi assay. The ethanol precipitation step ofthe Sampath-Reddi procedure is replaced by dialyzing (if the compositionis a solution) or diafiltering (if the composition is a suspension) thefraction to be assayed against water. The solution or suspension is thenequilibrated to 0.1% TFA. The resulting solution is added to 20 mg ofrat matrix. A mock rat matrix sample not treated with the protein servesas a control. This material is frozen and lyophilized and the resultingpowder enclosed in #5 gelatin capsules. The capsules are implantedsubcutaneously in the abdominal thoracic area of 21-49 day old male LongEvans rats. The implants are removed after 7-14 days. Half of eachimplant is used for alkaline phosphatase analysis [see, Reddi et al,Proc. Natl. Acad. Sci., 69:1601 (1972)].

The other half of each implant is fixed and processed for histologicalanalysis. 1 μm glycolmethacrylate sections are stained with Von Kossaand acid fuschin to score the amount of induced bone and cartilageformation present in each implant. The terms +1 through +5 represent thearea of each histological section of an implant occupied by new boneand/or cartilage cells and matrix. A score of +5 indicates that greaterthan 50% of the implant is new bone and/or cartilage produced as adirect result of protein in the implant. A score of +4, +3, +2, and +1would indicate that greater than 40%, 30%, 20% and 10% respectively ofthe implant contains new cartilage and/or bone.

The BMP-11 proteins of this invention may be assessed for activity onthis assay.

EXAMPLE 7

Biological Activity of Expressed BMP-11

To measure the biological activity of the expressed BMP-11 proteinsobtained in Example 3 above, the proteins are recovered from the cellculture and purified by isolating the BMP-11 proteins from otherproteinaceous materials with which they are co-produced as well as fromother contaminants. The purified-protein may be assayed in accordancewith the rat bone formation assay described in Example 6.

Purification is carried out using standard techniques known to thoseskilled in the art.

Protein analysis is conducted using standard techniques such as SDS-PAGEacrylamide [Laemmli, Nature. 227:680 (1970)] stained with silver[Oakley, et al. Anal. Biochem. 105:361 (1980)] and by immunoblot[Towbin, et al. Proc. Natl. Acad. Sci. USA 76:4350 (1979)].

The foregoing descriptions detail presently preferred embodiments of thepresent invention. Numerous modifications and variations in practicethereof are expected to occur to those skilled in the art uponconsideration of these descriptions. Those modifications and variationsare believed to be encompassed within the claims appended hereto.

EXAMPLE 8

Tests to Determine Activin Activity of BMP-11

Purification is carried out using standard techniques known to thoseskilled in the art. It is contemplated, as with other proteins of theTGF-β superfamily, that purification may include the use of Heparinsepharose.

Protein analysis is conducted using standard techniques such as SDS-PAGEacrylamide [Laemmli, Nature 227:680 (1970)] stained with silver [Oakley,et al. Anal. Biochem. 105:361 (1980)] and by immunoblot [Towbin, et al.Proc. Natl. Acad. Sci. USA 76:4350 (1979)].

BMP-11 proteins may be further characterized by their ability tomodulate the release of follicle stimulating hormone (FSH) inestablished in vitro bioassays using rat anterior pituitary cells asdescribed in, for example, Vale et al, Endocrinology, 91:562-572 (1972);Ling et al., Nature, 321:779-782 (1986) or Vale et al., Nature,321:776-779 (1986), the disclosures of which are hereby incorporated byreference. Alternatively, BMP-11 may be characterized by their abilityto stimulate erythropoietin activity in the human K-562 cell line, asdescribed by Lozzio et al., Blood, 45:321-334 (1975) and U.S. Pat. No.5,071,834, at column 15, the disclosures of which are herebyincorporated by reference.

In addition, BMP-11 may be characterized by their activity in cellsurvival assays, as described in Schubert, Nature, 344:868-870 (1990),the disclosure of which is incorporated by reference.

The foregoing descriptions, detail presently preferred embodiments ofthe present invention. Numerous modifications and variations in practicethereof are expected to occur to those skilled in the art uponconsideration of these descriptions. Those modifications and variationsare believed to be encompassed within the claims appended hereto.

1-27. (canceled)
 28. A purified BMP-11 polypeptide, wherein thepolypeptide comprises an amino acid sequence encoded by a DNA sequenceselected from the group consisting of: (a) nucleotides 1 to 759 of SEQID NO:10; (b) nucleotides encoding amino acids −253 to −1 of SEQ IDNO:11; and (c) a nucleotide sequence that hybridizes under stringentconditions with the complement of the nucleotide sequence of (a) or (b)wherein the stringent conditions comprise hybridizing at 65° C. andwashing at 65° C. in 0.1×SSC, 0.1% SDS.
 29. A purified BMP-11polypeptide encoded by a DNA sequence chosen from: (a) nucleotides 1 to759 of SEQ ID NO:10; (b) nucleotides encoding amino acids −253 to −1 ofSEQ ID NO:11; and (c) a nucleotide sequence that hybridizes understringent conditions with the complement of the nucleotide sequence of(a) or (b) wherein the stringent conditions comprise hybridizing at 65°C. and washing at 65° C. in 0.1×SSC, 0.1% SDS.
 30. The BMP-11polypeptide of claim 28, wherein the polypeptide comprises amino acids−253 to −1 of SEQ ID NO:11.
 31. The BMP-11 polypeptide of claim 29,wherein the DNA sequence is chosen from nucleotides 1 to 759 of SEQ IDNO:10 and nucleotides encoding amino acids −253 to −1 of SEQ ID NO:11.32. A pharmaceutical composition comprising an effective amount of theBMP-11 polypeptide of claim
 28. 33. The pharmaceutical composition ofclaim 32 in admixture with a pharmaceutically acceptable vehicle.
 34. Apharmaceutical composition comprising an effective amount of the BMP-11polypeptide of claim
 30. 35. The pharmaceutical composition of claim 34in admixture with a pharmaceutically acceptable vehicle.
 36. Thepharmaceutical composition of claim 31 in admixture with apharmaceutically acceptable vehicle.
 37. A method of inhibiting BMP-11activity in a mammal, comprising administering a pharmaceuticalcomposition of claim 32 to the mammal, thereby inhibiting a BMP-11activity.
 38. A method of promoting neuronal cell survival comprisingadministering to a mammal a pharmaceutical composition of claim
 32. 39.A method of promoting neuronal cell survival comprising administering toa mammal a pharmaceutical composition of claim
 33. 40. A method ofpromoting neuronal cell survival comprising administering to a mammal apharmaceutical composition of claim
 34. 41. A method of promotingneuronal cell survival comprising administering to a mammal apharmaceutical composition of claim
 35. 42. A method of promotingneuronal cell survival comprising administering to a mammal apharmaceutical composition of claim
 36. 43. A method of treating amammal, comprising administering a pharmaceutical composition of claim32 to a mammal to treat one or more conditions selected from bonedefects, connective tissue defects, periodontal disease, and tissuewounds.
 44. A pharmaceutical composition comprising the BMP-11polypeptide of claim 28 and a factor chosen from epidermal growth factor(EGF), fibroblast growth factor (FGF), platelet derived growth factor(PDGF), transforming growth factors (TGF-α and TGF-β), k-fibroblastgrowth factor (kFGF), parathyroid hormone (PTH), leukemia inhibitoryfactor (LIF/HILDA/DIA), and insulin-like growth factor (IGF), inadmixture with a pharmaceutically acceptable vehicle.